Studies on the bacterial plasmids of gram positive bacteria are of immense value in biology and medicine because of their involvement n the causation of many diseases, their role in bacterial resistance to a broad spectrum of antibiotics and their recently discovered role as indispensable tools in recombinant DNA research. Since bacterial plasmids are small DNA molecules capable of autonomous replication, they also provide simple model systems for biological studies. This proposal involves studies on the regulation of plasmid pT181 DNA replication in Staphylococcus aureus. pT181 is a 4.4 kilobase plasmid encoding resistance to tetracycline and has a copy number of 20 per chromosome. An in vitro replication system has been developed for this plasmid. A plasmid coded protein (the repC protein) has been shown to be required for pT181 replication. The structural gene for the repC protein and the origin of replication has been sequenced. This project involves isolation and purification of repC protein from cell extracts and studies on its function and site of action on the pT181 DNA. Experiments will be performed with the repC protein to determine if it is involved in the initiation, elongation and/or termination of pT181 replication. Available data indicate that the amount of the repC protein in cells may determine pT181 copy number. In addition, it is possible that the synthesis of the repC protein is autoregulated. In vitro transcription and translation studies will be performed to study the regulation of repC mRNA and protein synthesis. In vitro deletions of pT181 DNA will be made in the region known to contain the sequences involved in pT181 copy control. These delations will be checked for any increase in copy number. The eventual goal of this project would be to elucidate the regulation of the pT181 DNA replication and copy number. In addidion, a comparative study on the basic replication properties of a few small staphylococcal plasmids like pT181, pC194 and pSN2, would be made in order to determine whether similarity exists in DNA sequences at the origin of replication and their mode of replication. This might reveal any similar mechanism of replication of these small plasmids in this gram positive bacterium.